Supporting Information

Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions

Elisabeth Poehlera, Christin Herzoga, Carsten Lottera, Simon A. Pfeiffera, Daniel

Aignerb, Torsten Mayrb and Stefan Nagla,*

aInstitut für Analytische Chemie, Universität Leipzig, Linnéstr 3, 04103 Leipzig,

GermanybInstitut für Analytische Chemie und Lebensmittelchemie, Technische Universität

Graz, Stremayrgasse 9 / III, 8010 Graz, Österreich

*Corresponding author: Stefan Nagl

E-mail: nagl@chemie.uni-leipzig.de

Tel. +49 341 97 36066, Fax. +49 341 97 36115, http://www.uni-leipzig.de/~nagl/

S1 Materials and methods

S1.1. Chemicals

4-Acryloylmorpholine, olyethylenglycol-diacrylate (OEG-DA, MW 258 and 700), 2-

hydroxy-4´-(2-hydroxy)-2-methylpropiophenone, 3-(trimethoxysilyl)propyl

methacrylate (TPM), 2.2-dimethoxy-2-phenylacetophenone (DMPA, 99 %),

phosphate buffered saline (PBS, 150 mM), trichloromethane, sorbitan monolaurate

(Tween 20), ampicilin sodium salt, plasma from bovine, α-lactalbumin from bovine

milk and β-lactoglobulin B from bovine milk were purchased from Sigma-Aldrich

(Steinheim, Germany). H3BO3, H3PO3, CH3COOH, NaOH, HCl, H2SO4, NaHCO3 and

iso-propanol were acquired from Merck (Darmstadt, Germany). Methanol

(ROTISOLV ≥99.98 %, Ultra LC-MS) and formic acid (98 % p.a.) were purchased

from Carl Roth (Karlsruhe, Germany). Ofloxacin was aquired from Dr. Winzer

Pharma GmbH (Berlin, Germany). Carbonate buffer was made of 100 mM NaHCO3

and the pH was adjusted to 9 by titration with 0.1M HCl.

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2015

Britton-Robinson buffer (BRB) was made of 10 mM H3BO3, H3PO3 and CH3COOH

and the pH was adjusted by titration with 1 M NaOH solution and monitored using a

pH meter Lab 850 (SI Analytics, Mainz, Germany). Ampholyte pH 4-7 was acquired

from AppliChem (Darmstadt, Germany). N-(3-([N-(3-(methacryloylamino)propyl)

amino]sulfonyl)-2,6-diisopropylphenyl)-N’-(4-([N-(3-(methacryloylamino)propyl)amino]

sulfonyl)-2,6-diisopropylphenyl)-1-(4-methyl-1-piperazinyl)-6,7,12-trichloroperylene-

3,4,9,10-tetracarboxylic bisimide (Perylenebisimide, PBI) was synthesized by Daniel

Aigner according to Ref. S1.

S1.2. Microfluidic free-flow isoelectric focusing (µFFIEF)

neMESYS syringe pumps (cetoni, Korbussen, Germany) were used for flow control in

the separation bed. The electric field was induced with a HCP 35-6500 power supply

(FuG Elektronik, Rosenheim, Germany) in negative mode. For incoupling of the

electric field inside the microfluidic chip, platinum electrodes were inserted in the

electrode inlets (Figure 1g) and connected with the power supply. For the FFIEF the

analyte solution was injected through the center inlet of the microchip (flow rate:

0.5 µL∙min-1 for the proteins, 1.0 µL∙min-1 for the blood plasma and 1.5 µL∙min-1 for

the antibiotics). Next to the analyte the ampholyte flows (0.1 % ampholyte 4-7, 50

ppm (w/w) Tween 20, flow rate: 1.0 µL∙min-1), the sheath flows (10 mM BRB pH 10

and 20 mM H2SO4 pH 1.4 respectively, flow rate: 3.0 µL∙min-1 for the proteins and the

plasma and 2.0 µL∙min-1 for the antibiotics) and the electrode flows (flow rate:

5.0 µL∙min-1 for the proteins, 2.0 µL∙min-1 for the blood plasma and 3.0 µL∙min-1 for

the antibiotics) were applied. The cathodic flow consisted of 10 mM BRB pH 10

(8.05 mS∙cm-1) and the anodic flow of 20 mM H2SO4 (pH 1.4).

S2. Spectroscopic measurements and sensor characterization

S2.1. Spectroscopic measurements of the sensor matrix and the biomolecules

Absorbance and emission spectra were recorded with a UV-Vis spectrometer (P650

from Jasco, Gross-Umstadt, Germany) and a spectrofluorometer (FP-6200 from

Jasco, Gross-Umstadt, Germany). The measured spectra with the employed filters

and light sources in the IEF are shown in Figure S1.

Figure S1. Composite spectra of (a) the applied proteins α-lactalbumin and β-

lactoglobulin B (dashed blue line: absorption, straight blue line: emission intensity by

excitation at 266 nm, dark grey: emission filter, violet: laser), (b) bovine blood plasma

(dashed dark blue line: absorption, straight dark blue line: emission intensity by

excitation at 266 nm, dark grey: emission filter, violet: laser) and (c) the antibiotics

ofloxacin and ampicillin (dashed red line: absorption of ofloxacin, straight red line:

emission intensity of ofloxacin at 266 nm excitation, dashed brown line: absorption of

ampicillin, straight brown line: emission intensity of ampicillin at 266 nm excitation,

light grey: emission filter, violet: laser line).

The fluorescence emission of ofloxacin was much brighter than that of ampicillin after

excitation at 266 nm (Figure S1c) therefore we set the concentration of ampicillin

around four times higher than the concentration of ofloxacin in the IEF for

demonstration purposes.

S2.2. Characterization of the NIR fluorescent pH sensor

Absorbance and fluorescence of the NIR fluorescent sensor layer at pH 3 using 620

nm excitation (Figure S2) were recorded. At 266 nm excitation virtually no

fluorescence of the pH sensor was detected.

Figure S2. Spectra of the pH sensor layer and the employed LED light source and

filters (fluorescence of the pH sensor was excited at 620 nm and pH 3, dashed green

line: absorption, straight green line: emission).

The on-chip characterization of the sensor layer was performed via excitation with a

660 nm LED (M660L3, Thorlabs, 640 mW) which excites the NIR fluorescent pH

sensor at the isosbestic point and 2.5x Fluor objective (NA 0.12, Zeiss, Jena,

Germany) for Fig. S2a,c,d or a 20x UPlan FL N objective (Olympus, Hamburg,

Germany) for the fast observation of response times in Fig. 2b. The pH sensitivity

was examined with 10 mM BRBs from pH 2 to pH 10. The pKa was determined to be

5.91 ± 0.09 (Figure S3a). Therefore the pH sensitive region extended from around

pH 4.7 to pH 7.2. The response time of the pH sensor, which was measured with fast

changes in the flow solution between pH 3 and pH 10 was determined to be a few

seconds in both directions (t95 acidic alkaline 5.9 s, t95 alkaline acidic 7.1 s,

Figure S3b). Because of the covalent linking of the indicator, the sensor layer showed

a very high stability against a hydrodynamic flow at a flow rate of 25 µL∙min-1 over 30

minutes with an intensity loss of 3.74 ppm∙s-1 (Figure S3c). Applying constant LED

illumination for 30 minutes, the intensities decrease with a rate of 26.8 ppm∙s-1

(Figure S3d).

Figure S3. NIR fluorescent pH sensor characterization via 660 nm LED

excitation (1000 mA) at λexc 650/60 nm, λem 732/68 nm and a 2.5x microscope

objective (a, c, d) or a 20x objective (b). (a) pH-dependency of the

fluorescence intensity, (b) response time of the integrated sensor: red arrows:

change to pH 3, blue arrows: change to pH 10, (c) flow stability of the sensor

matrix at a flow rate of 0.45 mm∙s-1, (d) photostability at constant illumination

over 30 minutes.

S2.3. Characterization of the isoelectric focusing To show that the separation of the analytes was caused by the isoelectric

focussing and not by free-flow electrophoresis we took a macroscopic image of

the separation of the antibiotics with a colour CCD Camera (Figure S4). The

bands move almost perfectly parallel over several millimetres in the separation

bed which is strongly indicative of a steady-state IEF. The parallel movement

of the bands can also be seen in the supplementary video (SV1) that starts

with switching on the electric field, shows the formation of the bands and then

follows the bands along the separation bed to the outlet (SV1).

Figure S4. Macroscopic colour CCD image of the antibiotics separation.

S3. Mass spectrum analysis

Mass spectra were measured from the antibiotics mixture before the

separation and from fractions obtained from different outlets using a 6520

Agilent Q-TOF mass spectrometer (Agilent, Santa Clara, USA) with an ESI

source. ESI conditions were a temperature of 300 °C and a drying gas flow of

5 L∙min-1. The nebulizer was set to 20 psig and the ionization voltage was

4000 V. The sample solution was pumped into the source with a flow of

10 µL∙min-1 with a syringe pump PHD 22/2000 (Harvard Apparatus, Holliston,

USA) and a glass syringe (Hamilton Gastight 1725, 250 µL, Hamilton,

Bonaduz, Switzerland).

Table S1. Results of the mass spectra of different fractions collected at the

outlets

sample determined m/z [M+H]+ intensity (a.u.)

before separation

Ampicillin

Ofloxacin

350.117

362.150

279570

934277

Ampicillin fraction

Ampicillin

Ofloxacin

350.115

362.149

37363

5620

Ofloxacin fraction

Ampicillin

Ofloxacin

350.114

362.150

1512

225311

Supplementary References1 D. Aigner, S. M. Borisov, P. Petritsch and I. Klimant, Chem. Commun., 2013, 49, 2139.